viral-transgene-expression-rodent-hearts-assessment-cardiac

Viral Transgene Expression in Rodent Hearts and the Assessment of Cardiac Arrhythmia Risk

Heart disease is the leading cause of morbidity and mortality worldwide. Due to the ease of handling&#160;and abundance of transgenic strains, rodents ...

University of Ottawa Heart Institute

University of Ottawa

Intermittent fasting (IF), a periodic energy restriction, has been shown to provide health benefits equivalent to prolonged fasting or caloric restriction. However, our understanding of the underlying mechanisms of IF-mediated metabolic benefits is limited. Here we show that isocaloric IF improves metabolic homeostasis against diet-induced obesity and metabolic dysfunction primarily through adipose thermogenesis in mice. IF-induced metabolic benefits require fasting-mediated increases of vascular endothelial growth factor (VEGF) expression in white adipose tissue (WAT). Furthermore, periodic adipose-VEGF overexpression could recapitulate the metabolic improvement of IF in non-fasted animals. Importantly, fasting and adipose-VEGF induce alternative activation of adipose macrophage, which is critical for thermogenesis. Human adipose gene analysis further revealed a positive correlation of adipose VEGF-M2 macrophage-WAT browning axis. The present study uncovers the molecular mechanism of IF-mediated metabolic benefit and suggests that isocaloric IF can be a preventive and therapeutic approach against obesity and metabolic disorders.

Intermittent fasting promotes adipose thermogenesis and metabolic homeostasis via VEGF-mediated alternative activation of macrophage.

Intermittent fasting (IF) is an effective dietary intervention to counteract obesity-associated metabolic abnormalities. Previously, we and others have highlighted white adipose tissue (WAT) browning as the main underlying mechanism of IF-mediated metabolic benefits. However, whether IF retains its efficacy in different models, such as genetically obese/diabetic animals, is unknown. Here, leptin-deficient ob/ob mice were subjected to 16 weeks of isocaloric IF, and comprehensive metabolic phenotyping was conducted to assess the metabolic effects of IF. Unlike our previous study, isocaloric IF-subjected ob/ob animals failed to exhibit reduced body weight gain, lower fat mass, or decreased liver lipid accumulation. Moreover, isocaloric IF did not result in increased thermogenesis nor induce WAT browning in ob/ob mice. These findings indicate that isocaloric IF may not be an effective approach for regulating body weight in ob/ob animals, posing the possible limitations of IF to treat obesity. However, despite the lack of improvement in insulin sensitivity, isocaloric IF-subjected ob/ob animals displayed improved glucose tolerance as well as higher postprandial insulin level, with elevated incretin expression, suggesting that isocaloric IF is effective in improving nutrient-stimulated insulin secretion. Together, this study uncovers the insulinotropic effect of isocaloric IF, independent of adipose thermogenesis, which is potentially complementary for the treatment of type 2 diabetes.

Thermogenesis-independent metabolic benefits conferred by isocaloric intermittent fasting in ob/ob mice.

Stomach and intestinal stem cells are located in discrete niches called the isthmus and crypt, respectively. Recent studies have demonstrated a surprisingly conserved role for Wnt signaling in gastrointestinal development. Although intestinal stromal cells secrete Wnt ligands to promote stem cell renewal, the source of stomach Wnt ligands is still unclear. Here, by performing single cell analysis, we identify gastrointestinal stromal cell populations with transcriptome signatures that are conserved between the stomach and intestine. In close proximity to epithelial cells, these perictye-like cells highly express telocyte and pericyte markers as well as Wnt ligands, and they are enriched for Hh signaling. By analyzing mice activated for Hh signaling, we show a conserved mechanism of GLI2 activation of Wnt ligands. Moreover, genetic inhibition of Wnt secretion in perictye-like stromal cells or stromal cells more broadly demonstrates their essential roles in gastrointestinal regeneration and development, respectively, highlighting a redundancy in gastrointestinal stem cell niches.

Single cell and genetic analyses reveal conserved populations and signaling mechanisms of gastrointestinal stromal niches.

Cardiac hypertrophy is a key biological response to injurious stresses such as pressure overload and, when excessive, can lead to heart failure. Innate immune activation by danger signals, through intracellular pattern recognition receptors such as nucleotide-binding oligomerization domain 1 (Nod1) and its adaptor receptor-interacting protein 2 (RIP2), might play a major role in cardiac remodeling and progression to heart failure. We hypothesize that Nod1/RIP2 are major contributors to cardiac hypertrophy, but may not be sufficient to fully express the phenotype alone.

Innate Immune Nod1/RIP2 Signaling Is Essential for Cardiac Hypertrophy but Requires Mitochondrial Antiviral Signaling Protein for Signal Transductions and Energy Balance.

The global prevalence of metabolic disorders, such as obesity, diabetes and fatty liver disease, is dramatically increasing. Both genetic and environmental factors are well-known contributors to the development of these diseases and therefore, the study of epigenetics can provide additional mechanistic insight. Dietary interventions, including caloric restriction, intermittent fasting or time-restricted feeding, have shown promising improvements in patients' overall metabolic profiles (i.e., reduced body weight, improved glucose homeostasis), and an increasing number of studies have associated these beneficial effects with epigenetic alterations. In this article, we review epigenetic changes involved in both metabolic diseases and dietary interventions in primary metabolic tissues (i.e., adipose, liver, and pancreas) in hopes of elucidating potential biomarkers and therapeutic targets for disease prevention and treatment.

Understanding Dietary Intervention-Mediated Epigenetic Modifications in Metabolic Diseases.

Atrial Fibrillation (AF) is the most common supraventricular tachyarrhythmia that is typically associated with cardiovascular disease (CVD) and poor cardiovascular health. Paradoxically, endurance athletes are also at risk for AF. While it is well-established that persistent AF is associated with atrial fibrosis, hypertrophy and inflammation, intensely exercised mice showed similar adverse atrial changes and increased AF vulnerability, which required tumor necrosis factor (TNF) signaling, even though ventricular structure and function improved. To identify some of the molecular factors underlying the chamber-specific and TNF-dependent atrial changes induced by exercise, we performed transcriptome analyses of hearts from wild-type and TNF-knockout mice following exercise for 2 days, 2 or 6 weeks of exercise. Consistent with the central role of atrial stretch arising from elevated venous pressure in AF promotion, all 3 time points were associated with differential regulation of genes in atria linked to mechanosensing (focal adhesion kinase, integrins and cell-cell communications), extracellular matrix (ECM) and TNF pathways, with TNF appearing to play a permissive, rather than causal, role in gene changes. Importantly, mechanosensing/ECM genes were only enriched, along with tubulin- and hypertrophy-related genes after 2 days of exercise while being downregulated at 2 and 6 weeks, suggesting that early reactive strain-dependent remodeling with exercise yields to compensatory adjustments. Moreover, at the later time points, there was also downregulation of both collagen genes and genes involved in collagen turnover, a pattern mirroring aging-related fibrosis. By comparison, twofold fewer genes were differentially regulated in ventricles vs. atria, independently of TNF. Our findings reveal that exercise promotes TNF-dependent atrial transcriptome remodeling of ECM/mechanosensing pathways, consistent with increased preload and atrial stretch seen with exercise. We propose that similar preload-dependent mechanisms are responsible for atrial changes and AF in both CVD patients and athletes.

Transcriptomic Bioinformatic Analyses of Atria Uncover Involvement of Pathways Related to Strain and Post-translational Modification of Collagen in Increased Atrial Fibrillation Vulnerability in Intensely Exercised Mice.

Understanding the role of Iroquois homeobox transcription factor 5 (IRX5) in cardiac function: getting to the (human) heart of the matter.

Obesity is mainly due to excessive food intake. IRX3 and IRX5 have been suggested as determinants of obesity in connection with the intronic variants of FTO, but how these genes contribute to obesity via changes in food intake remains unclear. Here, we show that mice doubly heterozygous for Irx3 and Irx5 mutations exhibit lower food intake with enhanced hypothalamic leptin response. By lineage tracing and single-cell RNA sequencing using the Ins2-Cre system, we identify a previously unreported radial glia-like neural stem cell population with high Irx3 and Irx5 expression in early postnatal hypothalamus and demonstrate that reduced dosage of Irx3 and Irx5 promotes neurogenesis in postnatal hypothalamus leading to elevated numbers of leptin-sensing arcuate neurons. Furthermore, we find that mice with deletion of Irx3 in these cells also exhibit a similar food intake and hypothalamic phenotype. Our results illustrate that Irx3 and Irx5 play a regulatory role in hypothalamic postnatal neurogenesis and leptin response.

Irx3 and Irx5 in Ins2-Cre cells regulate hypothalamic postnatal neurogenesis and leptin response.

Ischemic heart disease is the leading cause of death in the United States, Canada, and worldwide. Severe disease is characterized by coronary artery occlusion, loss of blood flow to the myocardium, and necrosis of tissue, with subsequent remodeling of the heart wall, including fibrotic scarring. The current study aims to demonstrate the efficacy of quantitating infarct size via two-dimensional (2-D) echocardiographic akinetic length and four-dimensional (4-D) echocardiographic infarct volume and surface area as in vivo analysis techniques. We further describe and evaluate a new surface area strain analysis technique for estimating myocardial infarction (MI) size after ischemic injury. Experimental MI was induced in mice via left coronary artery ligation. Ejection fraction and infarct size were measured through 2-D and 4-D echocardiography. Infarct size established via histology was compared with ultrasound-based metrics via linear regression analysis. Two-dimensional echocardiographic akinetic length ( = 0.76,  = 0.03), 4-D echocardiographic infarct volume ( = 0.85,  = 0.008), and surface area ( = 0.90,  = 0.002) correlate well with histology. Although both 2-D and 4-D echocardiography were reliable measurement techniques to assess infarct, 4-D analysis is superior in assessing asymmetry of the left ventricle and the infarct. Strain analysis performed on 4-D data also provides additional infarct sizing techniques, which correlate with histology (surface strain:  = 0.94,  < 0.001, transmural thickness:  = 0.76,  = 0.001). Two-dimensional echocardiographic akinetic length, 4-D echocardiography ultrasound, and strain provide effective in vivo methods for measuring fibrotic scarring after MI. Our study supports that both 2-D and 4-D echocardiographic analysis techniques are reliable in quantifying infarct size though 4-D ultrasound provides a more holistic image of LV function and structure, especially after myocardial infarction. Furthermore, 4-D strain analysis correctly identifies infarct size and regional LV dysfunction after MI. Therefore, these techniques can improve functional insight into the impact of pharmacological interventions on the pathophysiology of cardiac disease.

Quantification of murine myocardial infarct size using 2-D and 4-D high-frequency ultrasound.

Previous studies have established that transmural gradients of the fast transient outward K current () correlate with regional differences in action potential (AP) profile and excitation-contraction coupling (ECC) with high  expression in the epimyocardium (EPI) being associated with short APs and low contractility and vice versa. Herein, we investigated the effects of altering the  gradients on transmural contractile properties using mice lacking  (Irx5-KO) or lacking  (K4.2-KO) or both. Irx5-KO mice exhibited decreased global LV contractility in association with elevated , as well as reduced cell shortening and Ca transient amplitudes in cardiomyocytes isolated from the endomyocardium (ENDO) but not in cardiomyocytes from the EPI. Transcriptional profiling revealed that the primary effect of  ablation on ECC-related genes was to increase  gene expression (i.e.,  and ) in the ENDO, but not the EPI. By contrast, K4.2-KO mice showed selective increases in cell shortening and Ca transients in isolated EPI cardiomyocytes, leading to enhanced ventricular contractility and mice lacking both  and  displayed elevated ventricular contractility, comparable to K4.2-KO mice, demonstrating a dominant role of -dependent modulation of  in the regulation of contractility. Our findings show that the transmural electromechanical heterogeneities in the healthy ventricles depend on the -dependent  gradients. These observations provide a useful framework for assessing the molecular mechanisms underlying the alterations in contractile heterogeneity seen in the diseased heart. Irx5 is a vital transcription factor that establishes the transmural heterogeneity of ventricular myocyte contractility, thereby ensuring proper contractile function in the healthy heart. Regional differences in excitation-contraction coupling in the ventricular myocardium are primarily mediated through the inverse relationship between Irx5 and the fast transient outward K current () across the ventricular wall.

Irx5 and transient outward K currents contribute to transmural contractile heterogeneities in the mouse ventricle.

Aberrant ketogenesis is correlated with the degree of steatosis in non-alcoholic fatty liver disease (NAFLD) patients, and an inborn error of ketogenesis (mitochondrial HMG-CoA synthase deficiency) is commonly associated with the development of the fatty liver. Here we aimed to determine the impact of Hmgcs2-mediated ketogenesis and its modulations on the development and treatment of fatty liver disease.

Article	Year
Intermittent fasting promotes adipose thermogenesis and metabolic homeostasis via VEGF-mediated alternative activation of macrophage. Cell research\| PubMed ID: 29039412	2017
Thermogenesis-independent metabolic benefits conferred by isocaloric intermittent fasting in ob/ob mice. Scientific reports\| PubMed ID: 30792482	2019
Single cell and genetic analyses reveal conserved populations and signaling mechanisms of gastrointestinal stromal niches. Nature communications\| PubMed ID: 31953387	2020
Innate Immune Nod1/RIP2 Signaling Is Essential for Cardiac Hypertrophy but Requires Mitochondrial Antiviral Signaling Protein for Signal Transductions and Energy Balance. Circulation\| PubMed ID: 33070627	2020
Understanding Dietary Intervention-Mediated Epigenetic Modifications in Metabolic Diseases. Frontiers in genetics\| PubMed ID: 33193730	2020
Transcriptomic Bioinformatic Analyses of Atria Uncover Involvement of Pathways Related to Strain and Post-translational Modification of Collagen in Increased Atrial Fibrillation Vulnerabilit... Frontiers in physiology\| PubMed ID: 33424629	2020
Understanding the role of Iroquois homeobox transcription factor 5 (IRX5) in cardiac function: getting to the (human) heart of the matter. Cardiovascular research\| PubMed ID: 33739382	2021
Irx3 and Irx5 in Ins2-Cre cells regulate hypothalamic postnatal neurogenesis and leptin response. Nature metabolism\| PubMed ID: 33859429	2021
Quantification of murine myocardial infarct size using 2-D and 4-D high-frequency ultrasound. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 34995167	2022
Irx5 and transient outward K currents contribute to transmural contractile heterogeneities in the mouse ventricle. American journal of physiology. Heart and circulatory physiology\| PubMed ID: 35245131	2022
Hmgcs2-mediated ketogenesis modulates high-fat diet-induced hepatosteatosis. Molecular metabolism\| PubMed ID: 35421611	2022

Kyoung-Han Kim

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